Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic painful bladder syndrome that affects the lives of approximately one million people in the United States. BPS/IC is also becoming increasingly important for the veterans population as it was reported in 2006 to be the fourth most common urologic disease as well as the primary diagnosis for 1.4% of all VA healthcare users (rate increased 13.7% and raw numbers increased 38% from 1999-2002, most recent data available). The etiology of BPS/IC is unknown, and there is currently no reliably effective treatment. It therefore continues to be important to study the pathogenesis of this debilitating disorder, in order to systematically devise effective, disease-specific therapies. Epithelial abnormalities are the most consistent findings in bladder biopsies from BPS/IC patients, including denudation, tears, or thinning of the epithelium, coupled with abnormal proteoglycan and protein expression (including tight junction and adherens proteins, vimentin, and uroplakin III). Explanted BPS/IC bladder epithelial cells also have decreased proliferation in vitro along with the same abnormal proteoglycan and protein expression as seen in patient bladder biopsies in vivo. In addition, BPS/IC cell explants secrete a Frizzled 8-related anti-proliferative factor (APF) (sialyl-Galb1-3GalNAcaO-Thr-Val-Pro-Ala-Ala-Val-Val-Val-Ala) that is not secreted by control cells, is found uniquely in urine from approximately 95% of BPS/IC patients (but not in control urine), and which induces the same abnormalities in proliferation and protein expression in normal bladder epithelial cells as seen in IC cells. The bladder pain plus increased urinary frequency and urgency experienced by BPS/IC patients may therefore result from aberrant bladder epithelial cell gene expression/differentiation that is unique to this syndrome and which results in bladder epithelial thinning or ulceration, leakiness, and abnormal cell protein expression. Because previous microarray experiments showed abnormal BPS/IC bladder epithelial cell expression of several differentiation proteins plus a chromatin remodeling protein, recent studies were performed which determined that BPS/IC cells indeed display an abnormal complement of certain gene regulatory proteins (including specific histone deacetylases (HDACs), transcription factors, and microRNAs) that could explain their abnormal gene expression and differentiation. Therefore, the proposed research will address the hypothesis that the gene expression abnormalities that we and others have described in BPS/IC bladder epithelial cells both in vivo and/or in vitro result from aberrant levels of specific gene regulatory factors. Our preliminary findings of abnormal HDACs, specific transcription factors, and microRNAs in explanted BPS/IC cells will be confirmed using explants from additional BPS/IC patients and controls, and the potential role for these and other regula- tory factors in abnormal specific gene expression determined using Western blot, chromatin immunoprecipita- tion (ChIP), microRNA microarray, and quantitative real-time polymerase chain reaction (qRT-PCR) techniques. The role of each regulatory factor in causing the BPS/IC cell phenotype will be confirmed via loss or gain of function experiments (including siRNA knockdown and transfection). Because APF can induce the same changes in proliferation and permeability, as well as many of the consistent gene expression abnormal- ities found in BPS/IC bladder cells in vivo and/or in vitro, the role of this toxin in regulation of these chromatin- related factors willalso be studied, and the ability of four APF inhibitors (shown previously to normalize proliferation, paracellular permeability, and tight junction protein expression in BPS/IC cells) to normalize chromatin regulatory factors in BPS/IC cells determined. Finally, the ability of a readily availabl, relatively nontoxic HDAC stimulator (theophylline) to normalize gene expression in BPS/IC cells will also be established. These studies should yield valuable information about the mechanism of aberrant gene expression/ differentiation in BPS/IC bladder cells, potentially leading to development of more effective therapies.